The objectives of this research are to devise improved non-viral gene carriers and to apply them in a Gene-Directed Enzyme Prodrug Therapy (GDEPT) protocol in a rat brain human tumor model. The question we will address is: will an optimized non-viral GDEPT protocol expressing yeast cytosine deaminase provide better therapy then a targeted liposome chemotherapy protocol in brain tumors? The hypothesis guiding this plan is that a combination of multiple levels of targeting will result in robust tumor-specific cytosine deaminase gene expression and improved tumor therapy. The four levels of targeting employed in these plans are: convective enhanced diffusion (CED) into the brain tumor, bioresponsive liposome gene formulations, CD44 receptor targeting, and a tumor matrix targeted enzyme. Tumor-localized cytosine deaminase will then convert the systemically administered prodrug, fluorocytosine (FC), to fluorouracil (FU), the active agent. This particular combination of approaches will enhance access of the carrier to the tumor, improve percolation of the carrier through the tumor, increase carrier attachment to tumor cells and improve intracellular delivery of the plasmid and bystander activity of the therapeutic gene. There are four major activities in the program: (1) synthesis of novel components and assembly of the components with plasmid DNA into a small diameter, stable liposome designated a nanolipoparticle (NLP)(2) characterization of the factors that control the performance of the NLP in cells (pH sensitivity, cell association, gene transfer activity, toxicity) and in animals (distribution, pharmacokinetics, toxicity and gene expression) after CED into the brain. (3) Construction of an extracellular matrix targeted cytosine deaminase to improve the bystander effect. (4) Comparison of the therapeutic effect of the optimized GDEPT to the effect of fluoroorotic acid delivered in a CD44 targeted liposome of similar physicochemical characteristics as the NLP. Fluorocytosine and fluoroorotic acid both exert their therapeutic effect through FU. The concentration of FU and metabolites will be measured in the tumor and plasma. We will determine which approach provides a greater drug concentration x time profile in the tumor as opposed to non-target cells. The combination of synthetic chemistry, molecular biology, pharmaceutics, and pharmacology will permit us to determine if a non-viral GDEPT is superior to a targeted prodrug for the treatment of brain tumors when both protocols are given by CED. These results will provide a rationale for targeted therapies for human brain tumors.
